CETP INHIBITOR POLYPEPTIDE, ANTIBODIES AGAINST THE SYNTHETIC POLYPEPTIDE AND PROPHYLACTIC AND THERAPEUTIC ANTI-ATHEROSCLEROSIS TREATMENTS

Abstract: A polypeptide and analogues thereof inhibit cholesteryl ester transfer protein (CETP). An anti-atherosclerosis composition comprises an anti-atherosclerosis effective amount of the polypeptide and a pharmaceutically acceptable carrier. An anti-atherosclerosis kit comprises in separate sterile containers at least one unit of the composition containing the polypeptide, one syringe and one needle. An antibody has specificity for the polypeptide of the invention, the baboon CETP 4kD polypeptide inhibitor, the 1-36 amino acid N-terminal fragment of apo C-I, modified apo A-I (MW:31kD) or modified apo E (MW:41kD). A method of preventing atherosclerosis in a mammal being predisposed to that condition comprises administering to the mammal a prophylactically effective amount of the polypeptide of the invention, and a method of treating a mammal afflicted with atherosclerosis comprises the administration of a therapeutically effective amount of the polypeptide disclosed herein.

Description: CETP INHIBITOR POLYPEPTIDE, ANTIBODIES AGAINST THE SYNTHETIC POLYPEPTIDE AND PROPHYLACTIC AND THERAPEUTIC ANTI-ATHEROSCLEROSIS TREATMENTS BACKGROUND OF THE INVENTION This application is a continuation-in-part of application, Serial No. 07/811,049, which was filed on December 18, 1991. The work leading to the present invention was partially supported by National Heart, Lung and Blood Institute Grants Nos. HL28972 and HL41256, and Contract No. HV53030. The government may hold rights in the present patent. Field of the Invention This invention relates to an endogenous baboon plasma cholesteryl esters transfer protein (CETP) inhibitor poiypeptide. More specifically, this invention relates to the identification and characterization of the poiypeptide and to novel synthetic peptides possessing inhibitory activity of CETP. The endogenous inhibitory peptide has a molecular weight of 4000, is present in plasma in the form of modified apo A-l and apo E having molecular weights of 31kD and 41kD, respectively, and has a common amino acid sequence with the N-terminal fragment of apo C-1. This invention also relates to an anti-atherosclerosis composition, a kit, and to antibodies raised against the N-terminal amino acid sequence of the inhibitory poiypeptide. The inhibitory peptide of the invention, fragments thereof and analogues thereof are useful for the prophylactic and therapeutic treatment of atherosclerosis. Description of the Background Atherosclerosis is one of the most widespread health problems in the United States today as are its attendant complications, particularly coronary heart disease. A number of risk factors have been associated with the development of premature atherosclerosis, primarily elevated plasma cholesterol levels. Due to the crucial role cholesterol appears to play in the occurrence of heart disease, a great deal of attention has been devoted to studying its synthesis, transport and metabolism in the human body. Of particular interest is the establishment of relationships between the levels of plasma lipoproteins or serum lipids and the risk of development of coronary heart disease. Both high density lipoproteins (HDL) and low density lipoproteins (LDL) carry cholesterol mainly in the form of cholesteryl esters (CE). There are some indications, however, that while LDL cholesterol is a positive risk factor, HDL cholesterol is an even more important negative risk factor. Although the exact functions of these lipoproteins have not been completely established, HDL appears to serve for the removal of cholesterol from peripheral cells and its transport back to the liver, where a large proportion of the cholesterol excreted from the body is removed. LDL and HDL are believed to play key roles in the development of cardiovascular disease by overloading the lysosomes of the walls of arterial cells with metabolites which are generally hydrolyzed slowly, such as CE and triglycerides. These products are evacuated from the liver and intestine by plasma LDL. When the amount of lipids to be transported exceeds the transporting capacity of HDL to the liver for excretion, CE become deposited in the cells in certain critical areas, such as arterial walls. This overloading eventually results in impaired cell function, and if continued may produce cell death. A continuous overloading results in the accumulation of cellular debris and the formation of atherosclerotic plaque in the vessel wall. This, in turn, leads to the blockage of the affected artery and/or muscular spasm, events which may manifest themselves as coronary heart disease or strokes. Thus, the level of HDL in plasma has been negatively correlated with the probability of developing atherosclerosis in humans and experimental animals. Although the level of HDL has been shown to vary considerably among individuals, the means of regulation of such plasma level remains to be elucidated. CETP transfers CE from HDL to VLDL and LDL, and it has been suggested that it plays an important role in the regulation of plasma HDL levels. Some hyperalphahpoproteinemic patients were reported to have high levels of large HDL particles that were clearly separate from LDL. Plasma samples from these patients were shown SUBSTITUTE SHEET (RULE 2β) to lack CETP activity (Koizumi et al, Atherosclerosis 58:175-186 (1985)). A homozygous subject with familial hj eralphalipoproteinemia was found to have impaired transfer of CE from HDL to LDL (Yokoyama et al, Artery 14:43-51 (1986)). A fraction of density d>1.21 g/ml from the subject's plasma evidenced substantial CETP activity with normal HDL. The HDL, however, proved to be a poor substrate for CETP. Certain animal sires and their progeny possess unusual lipoproteins patterns, e.g., lipoproteins of a density intermediate to that of LDL and HDL, or large high density lipoproteins. These lipoproteins have been designated HDL j and the animal phenotype as "high HDLi". Baboon strains possessing, for instance, patterns of either high or low HDL X are known. In most cases, HDL X separates either as a distinct peak between LDL and HDL or as a shoulder to the HDL peak, and is induced by a high cholesterol, high lard (HCHF) diet. The proportion of HDL j diminishes when the baboons are fed a diet that is either enriched in polyunsaturated fat, with or without cholesterol. Occasionally, however, the amount of HDL j present in high HDL X baboons fed the chow diet is low. In some baboon families, the level of plasma HDL X was shown to increase when the animals are challenged with a HCHF diet. When fed a HCHF diet, the baboons also show higher plasma HDL. More generally, the accumulation of HDL in baboons as well as in humans is associated with a slower transfer of CE from HDL as very low density lipoproteins (VLDL) and LDL. Thus, baboons with high HDL ! plasma levels are excellent as animal models for the study of hyperalphalipoproteinemia. In a previous study, some of the present inventors reported that a slower transfer of CE from HDL to VLDL and LDL was observed in high HDL t baboons. This was attributed to the presence of a CETP protein inhibitor associated with HDL and intermediate density lipoprotein (IDL) particles (Kushwaha et al, J.P. Lipid Res. 31:965-974 (1990)). An accumulation of ΗD ^ in the high HDL j baboons fed a HCHF diet was reported along with a slower transfer of CE from HDL to LDL. A similar protein was found in human plasma by Son and Zilversmit (Son and Zilversmit, B.B.A. 795:473- 480 (1984)). The human protein has a molecular weight of 31,000 and suppresses the transfer of triacylglycerol and CE. Several other species including rat, pig, and dog have been reported to readily accumulate HDL j in plasma. Kurasawa et al. (1985), supra, reported that a homozygous subject with familial hyperalphalipoproteinemia has impaired CE transfer between HDL and LDL (Kurasawa et al, J.B. Biochem. 98:1499-1408 (1985)). Separately, Yokoyama, et al. reported that a plasma fraction of d>1.21 g/ml of the same subject evidenced substantial CE transfer activity when tested with normal HDL (Yokoyama et al, Artery 14(1):43-51 (1986)). The HDL particles accumulated by this subject were substantially larger in molecular size than ordinary HDL 2 . HDL is generally divided into subfractions based on their particle sizes and densities. These fractions include HDL j , HDL 2 , and HDL 3 . HDL X has the largest particles and is usually not present in the plasma of normal humans or non-human primates. HDL 2 and HDL 3 are the normal components of human plasma. HDL 2 is larger than HDL 3 and differs between men and women. Many attempts have been made to interfere with the transport and transfer of cholesterol in mammalians in order to alter its plasma levels. Among them are the following. U.S. Patent No. 4,987,151 to Taboc discloses triterpene derivatives that inhibit acyl coenzyme Axholesteral acyltransferase (ACAT) enzyme. The ACAT is a cellular enzyme that is not present in plasma, and esterfies cellular cholesterol to form CE. This enzyme is different from the CE transfer protein (CETP) present in plasma. The CETP does not form CE as does the ACAT enzyme. Instead, the CETP transfers CE amongst different plasma lipoproteins. U.S. Patent No. 4,643,988 to Segrest, et al discloses amphipathic peptides which are capable of substituting for apo A-I in HDL. Apo A-I is known to stimulate the lecithin cholestero acyl transferase (LCAT) enzyme, a plasma enzyme that forms CE in HDL. Plasma CETP, in contradistinction, transfers CE from HDL to VLDL and LDL. The function of the CETP enzyme is, therefore, different from that of the LCAT enzyme, as well. The amino acid sequence of the Segrest et al peptides are, in addition, different from the sequences of the CETP inhibitor of this invention. Summary of the Invention This invention relates to a substantially pure poiypeptide having activity inhibitory of CE transfer protein (CETP). This invention also relates to an anti-atherosclerosis composition, comprising an anti- atherosclerosis effective amount of the poiypeptide described above; and a pharmaceutically-acceptable carrier. In addition, this invention relates to an anti-atherosclerosis kit, comprising in separate containers at least one unit of the composition described above; at least one syringe; and at least one needle. This invention also relates to an antibody having specificity for a poiypeptide selected from the group consisting of the poiypeptide described above; baboon CETP poiypeptide inhibitor; 1-36 amino acid N-terminal fragment of apo C-I; modified apo A-I (NW:31kD); modified apo E (MW:41D). In a different aspect, this invention relates to a method of preventing atherosclerosis in a mammal being predisposed to that condition, comprising administering to the mammal a prophylactically effective amount of the poiypeptide described above. This invention also relates to a method of treating a mammal afflicted with atherosclerosis comprising administering to the mammal a therapeutically effective amount of the poiypeptide described above. Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following discussion. Description of the Preferred Embodiment This invention arose from the desire by the inventors to provide a novel and unobvious approach to the prevention and treatment of atherosclerosis in humans. A schematic of what is known about the association of cholesterol with the different fractions of lipoproteins in plasma and in the liver is shown below: The metabolic steps leading to the accumulation ( ) of HDL 1; VLDL and LDL are shown above. HDL 2+3 collects cholesterol from extrahepatic cells, which is then esterified by LCAT to form cholesteryl esters (CE) and stored in the core of the particles. The HDL becomes larger in size (HDL j ) and may pick up apo E to attain a particle which is removed from LDL receptors (LDL-R) on liver cells. The CE enriched HDL X may also donate CE to VLDL and LDL. This is mediated by CETP. Due to the presence of the CETP inhibitor, such as the one provided herein, CE transfer is slow (bar) and the reciprocal transfer of triglycerides (TG) does not take place (X). The triglyceride-poor HDL is thus not a suitable substrate for hepatic triglyceride lipase (HTGL). Due to the presence of the CETP inhibitor, in plasma, VLDL and LDL are thus not available to the liver. As a consequence of this, the liver then increases the expression of messages for the increased production of LDL receptor and 3 hydroxy, methyl, glutaryl-coenzyme A (HMG-COA) synthase. The increase in LDL receptor in the liver leads to an increase in uptake of LDL or HDL, with apo E, and consequently, to a greater delivery of cholesterol to the liver. An increase in synthesis of HMG- CoA synthase leads to an increase in synthesis of cholesterol in the liver to meet all cellular needs. Thus, the presence of a CETP inhibitor in plasma will prevent the uptake of VLDL and/or LDL by tissues as well as the deposition of cholesteryl esters. In general, high levels of HDL have an anti-atherosderogenic effect whereas high levels of LDL have an atherogenic effect. The circulation in blood of compounds, such as cholesterol, that are insoluble in water requires the formation of particles. The insoluble components, e.g., cholesteryl esters and triglycerides, are packed in the core of the partides and surrounded by polar components such as proteins, phospholipids, and the like. These particles are called lipoproteins, and have, thus, an outer polar shell and a non-polar core. These assodations of lipoproteins containing CE in the core and, depending on their sizes and densities, have been named VLDL, IDL, LDL, and HDL. VLDL is the largest lipoprotein secreted by the Hver and is converted into IDL, and then to LDL, after the triglycerides contained by the VLDL are hydrolzyed by the lipoprotein lipase enzyme present on the surface of the arterial walls. LDL is the major lipoprotein that provides cholesteryl ester to extrapepatic and hepatic tissues. HDL is also secreted by the hver and is divided in HDL 2 and HDL 3 on the basis of size and density. A function of HDL is to pick up cholesterol from extrahepatic cells and deliver it to the Hver, either through VLDL and LDL or through large HDL which is enriched with apo E. These large HDL partides are called HDL : and they do not stay in the plasma for long periods of time. They are rapidly removed by the Hver or converted back to HDL 2 after donating their cholesteryl esters to VLDL and LDL. HDL X is not present in either normal humans or non-human primates. As indicated above, HDL ! appears as a distinct band in the plasma of baboons that have been fed a HCHF diet. From what is known, when cholesterol enters the blood stream it becomes assodated with HDL in the form of a CE, with the help of the LCAT enzyme. In HCHF-fed baboons, this appears as an HDL r CE fraction. The CE is then transferred from HDL to VLDL and LDL to form VLDL-CE and LDL-CE with the aid of the CETP enzyme. These partides then enter the Hver ceUs through the LDL receptor (LDL-R). After being metabolized in the Hver ceUs, the VLDL-CE is returned to the plasma and thus to the periphery of the mammalian body, where its deposition may occur leading to atherosclerosis. An inhibitor of CETP, such as the one provided by this invention, blocks the transfer of CE from HDL to VLDL and LDL. Instead, a shunt is favored that leads to the association of the CE with apo E and to the formation of HDL j -CE-apo E particles that can enter the Hver ceUs through the LDL receptor (LDL-R). The apoHpoprotein C-I of various spedes but not baboon, are known. The Apo C-I is a single poiypeptide of molecular weight 6600, consisting of 57 amino adds. It is a basic protein that is mainly present in VLDL and HDL, with HDL serving as a reservoir for this protein. LDL, on the other hand, contains Httle apo C-I. It has recently been shown that apo C-I displaces apo E from VLDL and affects its binding to the LDL receptor. The poiypeptide inhibitor of CETP that is described herein has a common sequence with the N-terminal fragment of apo C-I. This fragment indudes at least 36 amino acids as shown below. The endogenous poiypeptide (SE . ID. NO: 1) provided by this invention has a molecular weight of about 4,000 and becomes assodated or binds to apo A-I and apo E in plasma. Its N-terminal 36 amino adds are shown below. A poiypeptide having the sequence corresponding to amino adds 1 to 36 of the foUowing sequence (SEQ. ID. NO: 1) was synthesized by the inventors and shown to be inhibitory of CETP in vitro. The peptide has the foUowing sequence. 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile 25 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO: 1). Fragments of that poiypeptide (SEQ. ID. NO: 1) comprising the C-terminal fragments of amino acids 28 to 36 and amino adds 16 to 36 showed Hmited inhibitory activity of CETP at 50 μg. However, the fragment comprising the C-terminal amino adds 28 to 36 showed at 200 μg an activity inhibitory of CETP approximately the same as that of the 36 amino add peptide. The N-terminal fragments comprising amino acids 1 to 15, amino adds 1 to 20 and amino adds 1 to 10, as weU as the intermediate fragments comprising amino adds 15 to 30 and the like, corresponding to the synthesized peptide have been shown active as inhibitors of CETP. Polypeptides having the sequences corresponding to the foUowing two sequences designated (SEQ. ID. NO:2) and (SEQ. ID. NO:3) have also been synthesized by the inventors and shown to be inhibitors of CETP in vitro. The first of the foUowing two sequences (SEQ. ID. NO:2) is a baboon sequence like (SEQ. ID. NO:l) except that it has two additional amino adds at the beginning of the peptide. The second of the foUowing two sequences (SEQ. ID. NO:3) is a human sequence and varies from (SEQ. ID. NO:2) in seven of the thirty-eight amino adds in the sequence. Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-Gly- Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile-Asn-Arg-Ile-Lys-Gln- Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO:2) Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-Gly- Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu-Leu-Ile-Ser-Arg-Ile-Lys-Gln- Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO:3)- Analogues of the poiypeptide of the invention and fragments thereof having inhibitory activity of CETP are also part of the invention. The analogues may have one or more substitutions in their sequences while stiU preserving their inhibitory activity. Examples of analogues suitable as inhibitors of CETP are analogues of the peptide of the invention and fragments thereof, such as those where one or more of the amino acids are substituted in accordance with the guidelines provided below. The substitute amino acids may be selected from the group consisting of Glu, C α -methylAsp, and β-carboxyAsp for Asp; isoVal, norVal, Leu, and C α -methylVal for Val; Gly, β-Ala, C α -methylAla, and 2-amino butyric add for Ala; norLeu, isoLeu, and C α -methylLeu for Leu; ornithine, Arg, dtrulHne and C α -methylLys for Lys; Ala and 2-amino isobutyric acid for Gly; Gin, citruUine, and C α -methylAsn for Asn; P-Benzoyl Phe, Arg, and C α -methylTrp for Trp; 2 -amino adipic add, Asp, and C α -methylGlu for Glu; Leu, norLeu, and C α -methyllle for He; Lys, homoArg, dtrulHne and C α -methylArg for Arg; Asn, dtruUine, and C α -methylGln for Gin; 2-amino-4-phenylbutyric add, Leu and C α -methylPhe for Phe; Ser, Met and C α -methylThr for Thr; Thr and C α -methylSer for Ser; and 3,4-DehydroPro, Ser and C α -methylPro for Pro; and combinations thereof. However, other substitutions of a nature equivalent to that of the substituted amino add as is known in the art may also be utilized, alone or in combination with other substituents. It is therefore provided in accordance with this invention, a substantiaUy pure poiypeptide having activity inhibitory of CETP. In one embodiment of the invention, the poiypeptide is capable of inhibiting the binding of an about 31kD modified apo A-I poiypeptide present in the plasma of high HDL j baboons or a peptide of the sequence. 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 25 26 2728 29 30 31 32 33 34 35 36 Asn-Arg-Ile-Lys-GHi-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID.NO.1), to an antibody raised against the above peptide. In another embodiment, the poiypeptide of the invention is capable of inhibiting the binding of an about 4kD CETP inhibitor poiypeptide present in the plasma of high HDL j baboons to an antibody raised against a peptide of the formula 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 25 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO.1). In still another embodiment, the poiypeptide of this invention is capable of inhibiting the binding of an about 41kD modified apo E poiypeptide present in the plasma of high HDL j baboons with antibody raised against the 36 amino acid N-terminal fragment of apo C-I or a peptide of the formula 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 25 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-He-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO.1). In still another embodiment, the poiypeptide is capable of inhibiting the binding of the 36 amino add N-teπninal fragment of apo C-I or a peptide of the formula 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 25 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO.1), with an antibody raised against modified apo A-I. Preferred polypeptides are the polypeptides of the sequence 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 25 26 2728 29 30 31 32 33 34 35 36 Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO.1), Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-Gly- Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile-Asn-Arg-Ile-Lys-Gln- Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO:2), and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-Gly- Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu-Leu-He-Ser-Arg-fle-Lys-Gln- Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO:3); anti-4kD peptide antibody-binding inhibitory fragments of (SEQ. ID NO.: 1) thereof; and anti-4kD peptide antibody-binding inhibitory analogues of (SEQ. ID NO:l) thereof having at least one substitute amino add selected from the group consisting of Glu, C α -methylAsp, and β-carboxy Asp for Asp; isoVal, norVal, Leu, and C α -methylVal for Val; Gly, β-Ala, C α -methylAla, and 2-amino butyric acid for Ala; norLeu, isoLeu, and C α -methylLeu for Leu; ornithine, Arg, dtrulHne and C α -methylLys for Lys; Ala and 2-amino isobutyric acid for Gly; Gin, dtrulHne, and C α -methylAsn for Asn; P-BenzolyPhe, Arg, and C α -methyl Trp for Trp; 2-amino adipic acid, Asp, and C α -methylGlu for Glu; 2-amino adipic acid, Asp, and C α -methylTrp for Trp; Leu, norLeu, and C α -methyllle for lie; Lys, homoArg, dtrulHne and C α -methylArg for Arg; Asn, dtrulHne, and C α -methylGln for Gin; 2-amino-4-phenylbutyric acid, Leu and C α -methylPhe for Phe; Ser, Met and C α -methylThr for Thr; Thr and C α -methylSer for Ser; 3,4-DehydroPro, Ser and C α -methylPro for Pro; and combinations thereof. In one particular preferred embodiment, the poiypeptide contains amino adds 1 through 36 of the above sequence (SEQ. ID. NO. 1). In stiU another particularly preferred embodiment, the peptide is selected from the group consisting of peptide fragments (SEQ. ID. NO. 1) comprising amino acids 1 to 17, 1 to 20 and 1 to 25, and fragments thereof having anti-4kD peptide antibody/1-36 amino add peptide binding inhibitory activity. In stiU another preferred embodiment, the peptide fragments are selected from the group consisting of peptides comprising amino adds 1 to 18, and 1 to 28 of (SEQ. ID. NO. 1), and fragments thereof having anti-4kD peptide antibody/1-36 amino adds peptide binding inhibitory activity. Also preferred are analogues (SEQ. ID. NO. 1) with the Lys, Asp and Asn amino adds substituting for the Arg, Glu, and Gin amino adds; the Ser, Leu, and Ala amino acids substituting from the Thr, lie and Gly amino acids; the ornithine, dtrulHne and a aminoadipic add amino adds substituting for the Lys and Glu amino adds. Also preferred are the foUowing analogues (SEQ. ID. NO. 1). Peptides comprising amino add sequences where amide bond(s) (e.g., -C(O)-NH-) linking any pair, and up to aU pairs, of amino acids comprising amino acids 1 to 17, 1 to 20, 1 to 25, 1 to 36, and fragments thereof having anti-4kD peptide antibody/1-36 amino acid peptide binding inhibitory activity are replaced by thioether bonds (e.g., -CH 2 -S-) alkyl such as ethyl (-CH 2 -CH 2 -), and/or amino (e.g., - CH 2 -NH 2 -) linkages. These analogues may be purchased commerdaUy or prepared by methods know to those skilled in the art as long as the antibody 1-36 amino add peptide binding and CETP inhibitory activities of the peptides (analogues) are retained to some degree. However, other analogues (SEQ. ID. NO. 1) are also part of this invention as long as they preserve the inhibitory activity of the antibody/1-36 amino add peptide binding. The CETP inhibitory poiypeptide of the invention may be provided as a powder, preferably in freeze-dried form, as a solution, preferably frozen at below -20°C, and the like, to prevent proteolysis. This invention also provides an anti-atherosderosis composition, comprising an anti-atherosclerosis effective amount of the poiypeptide of the invention; and a pharmaceuticaUy-acceptable carrier. When the composition is used as preventative tool, it may contain 10 to 200 mg and more preferably 20 to 100 mg of the poiypeptide. However, other amounts are also suitable. When the composition is intended for therapeutic use, the amount of poiypeptide present is preferably about 10 to 400 mg, and more preferably about 20 to 300 mg. However, other amounts may also be utilized. Any and aU pharmaceuticaUy-acceptable carriers known in the art for administration of peptides to mammals, and preferably to humans, are suitable for use herein. These are known in the art and need not be further described herein. Examples, however, are saline, human serum albumin and starch. However, others may also be utilized. The composition of this invention may be provided in unit form, preferably in a sterile, closed container, and more preferably in a sealed container. A kit, comprising in separate containers at least one unit of the anti-atherosderosis composition of the invention; at least one syringe; and at least one needle. TypicaUy, a kit may contain from about 1 to 20 units of the composition of the invention, but could contain 50 units or more. In addition, the kit may contain 1 to 20, but and sometimes 50 or more syringes if they are disposable, and 1 to 20, but sometimes up to 50 or more needles if they are disposable. The components of the kit are provided in a sterile form, be it wrapped in a sealed, sterilized wrapping, or in some other way. If not disposable, the syringe and needle may be autodaved between uses. The composition of the invention is preferably administered intravenously, although it may also be administered intraperitoneaUy, subcutaneously or intramuscularly. The oral route is not permissible since the poiypeptide would be degraded in the addic pH of the stomach. The composition may preferably have a pH of about 7 to 9, and more preferably about 8 to 9, which may be adjusted with the addition of a base, add or buffer as is known in the art. This invention also provides an antibody having specificity for a poiypeptide selected from the group consisting of the polypeptides of the invention; the baboon CETP poiypeptide inhibitor, fragments thereof and analogues thereof; the 1-36 amino add N-terminal fragment of apo C-1; modified apo A-I (MW:31kD); and modified apo E (MW:41kD). The antibodies of the invention may be raised in mammals as is known in the art. (Albers, J.J. Hazzard, W.R., Immunochemical Quantification of the Human Lp(a) Lipoprotein, Lipids 9:15-26 (1974)). TypicaUy, the antibodies may be raised in rabbit, goat, sheep, pig, and chicken. However, other mammals may also be utilized. Preferred are rabbit antibodies. Also preferred are polychonal antibodies. However, monoclonal antibodies may also be prepared by methods known in the art (Kohler, G., and MUstein, D., Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity Nature (London) 256:495-497 (1975)). In one preferred embodiment, the antibody of the invention is capable of specifically binding to modified apo A-I. In another preferred embodiment, the antibody is capable of specificaUy binding to the baboon CETP inhibitor polypeptides of this invention. In another preferred embodiment, the antibody of the invention is capable of specificaUy binding to the poiypeptide of the sequence. 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 25 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-lle-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr fragments thereof and analogues thereof as described above. In stiU another preferred embodiment of the invention, the antibody is capable of specificaUy binding to modified apo E. The antibody of the invention, in another preferred embodiment is also capable of specificaUy binding to apo C-1, and more preferably to the 1-36 N-terminal fragment thereof. In another aspect of the invention, a method is provided for preventing atherosderosis in a mammal being predisposed to that condition. The method comprises administering to the mammal a prophylacticaUy effective amount of the poiypeptide of the invention, or of the anti-atherosderosis composition described above. In a preferred embodiment, the poiypeptide is administered in an amount of about 2 to 100 mg for preventative appHcations. However, other amounts may also be administered. The poiypeptide or composition thereof may be administered in a small volume of carrier, e.g., 0.2 to 1.5 ml of saline or other carriers, as is known in the art. The poiypeptide of the inventions may be administered intravenously, to a fragment of the population, particularly the human population, that is not afflicted by high blood cholesterol and hyperbetaHpoproteinemia, but, as detennined by other means, may be at risk of being afflicted by atherosclerosis. One such example may be a familial trait having been determined. The poiypeptide of this invention may be administered on a daUy basis, or at longer intervals if provided as a slow release composition as is known in the art, such as depoestradiol provided by the UpJohn Co. (UpJohn Co., Kalamazoo, MI). In another aspect, the present invention provides a method of treating a mammal afflicted with atherosclerosis. The method comprises administering to the mammal a therapeuticaUy affective amount of the poiypeptide of the invention. When administered for therapeutic purposes, the poiypeptide may be injected in an amount of about 10 to 400 mg, and more preferably 20 to 300 mg. However, other amounts as assessed by a practitioner in specific cases, may also be administered. In this case, as in the case of the prophylactic administration, the polypepti.de may be administered intravenously, among other routes. Having now generaUy described this invention, the same wiU be better understood by reference to certain specific examples, which are induded herein for purposes of iUustration only and are not intended to Hmiting of the invention or any embodiment thereof, unless so specified. Examples .vam lft 1 : Animals and Diet Adult male and female baboons (papio sp.) held in a baboon colony at the Southwest Foundation for Biomedical Research in San Antonio, Texas, were used as blood donors for these studies. Among these, 24 baboons had a high HDLj phenotype and 32 had a low HDL j phenotype (WiUiams, M.C., et al., Detection of Abnormal Lipoprotein In a Large Colony of Pedigreed Baboons Using High- Performance Gel Exclusion Chromatography, J. Chromat. 308:101- 109 (1984)). Half of the high HDL j baboons (n=16) were maintained on a HCHF diet, the composition of which has been previously described (Kushwaha, R.S., et al., Metabolism of Apolipoprotein B. In Baboons with Low and High Levels of Low Density Lipoprotein. J. Lipid Res. 27:497-507 (1986)). Most of the baboon donors of the low HDL j phenotype were maintained on a chow diet (Purina Monkey Chow, manufactured by Ralston Purina Co., St. Louis, Missouri). The monkey chow is low in fat (10% of total calories) and high in carbohydrate (62% of total calories). In addition, the chow has a very low cholesterol content (0.03 mg Kcal). The high HDL j baboons were progeny of two sires (X1672 and X102) who had a high HDL j phenotype. The low HDL j baboons were progeny of a number of sires who did not have a high HDL j phenotype. The presence of HDL X was detected by high performance Hquid chromatography (HPLC) as described previously (WilHams, et al., Detection of Abnormal Lipoprotein in a Large Colony of Pedigreed Baboons Using High-Performance Gel Exclusion Chromatography, J. Chromatography 308:101-109 (1984)). Example 2: Preparation of 3 H Cholesteryl Ester HDL High and low HDL j baboons were immobilized with 10 mg/kg of ketamine HC j and bled. The blood was coUected in tubes containing 1 mg/ml EDTA, and plasma separated by low speed centrifugation at 6°C. The plasma was treated with sodium azide, choloramphenicol, gentamycin sulfate, phenylmethyl-sulfonyl fluoride and DTNB as described previously (Kushwaha, R.S., et al., Impaired Plasma CE Transfer with Accumulation of Larger High Density Lipoproteins in Some Families of Baboons (papio sp.), J. Lipid Res. 31:965-973 (1990)). 20 to 60 μCi of tritiated cholesteryl linoleate were dissolved in ethanol and then added to the plasma. The plasma was flushed with nitrogen and incubated for 20 hours at 4°C. After incubation, HDL 3 was isolated by density gradient ultracentrifugation (McGiU, H.C. et al., Dietary Effects on Serum Lipoprotein of Dsylipoproteinemic Baboons with High HDL., Atheriosderosis 6:651-663 (1986); Redgrave, T.G., et al., Separation of Plasma Lipoprotein by Density-Gradient Ultracentrifugation, Ana.Biochem. 65:42-49 (1975)), dialyzed against saHne/EDTA, and used as a substrate for the CE transfer reaction. The total and free cholesterol contents of HDL 3 were measured prior to use in the assay. Example 3; Preparation of Acceptor Lipoproteins and CETP Source VLDL+LDL from low HDL j baboons was used as the acceptor of CE from HDL 3 . A VLDL+LDL fraction of d<1.040 g/ml was isolated from 100-200 ml of blood by sequential ultracentrifugation as described previously (Kushwaha, et al. (1986), supra). Total and free cholesterol contents in acceptor Hpoprotein were measured by enzymatic methods (Wako Pure Chemical Co.) (AUain, CC, et al., Enzymatic Determination of Total Serum Cholesterol Clin. Chem. 20:470-475 (1974)). After separation of VLDL+LDL, the bottom fraction was adjusted to d=1.21 g/ml of adding soHd KBr, and total Hpoprotein were isolated by ultracentrifugation (Kushwaha, et al. (1986), supra). The bottom fraction of d>1.21 g/ml was also coUected. AU Hpoprotein fractions and the Hpoprotein-deficient fraction of d>1.21 g/ml (LPDS) were dialyzed against saline/EDTA. The LPDS was used as the source of CETP. Fgflm le A: Cholesteryl Ester Transfer Assay The CE transfer activity of a sample was assayed by a modification of a procedure described previously (Kushwaha, et al., (1986), surpa). Briefly, 3 H CE-labeled HDL 3 containing 50-100 μg of CE from low HDL ! baboons was incubated with VLDL+LDL containing 100- 300 μg CE in the presence of LPDS. The acceptor Hpoprotein and the LPDS were obtained from low HDL j baboon plasma (chow diet). In some cases, the HDL 3 was obtained from high HDL j baboons maintained on the HCHF diet. The incubations were carried out at 4°C (control) and 37°C for 4-6 hrs. and terminated by placing the samples on ice. The assay mixture was then ultracentrifuged to separate VLDL+LDL having a d>1.040 g/ml, and the radioactivity in the Hpoprotein was counted as described previously (Kushwaha, et al. (1986), supra). Any difference observed in the radioactivity transferred from HDL to VLDL+LDL at 4°C and 37°C was attributed to CETP activity in the LPDS. Time course experiments gave a linear response up to 7 hrs. SimUarly, the CETP activity was linear with increasing LDPS, up to 140 μl LPDS, which was derived from an equivalent volume of plasma. They poiypeptide of the invention, and synthetic fragments thereof were added to the reaction mixture to determine their CETP inhibitory activities, along with an appropriate synthetic control peptide. The percent difference between the control experiment and the assay with inhibitor peptide was expressed as the inhibitor activity. Example 5; Identification of Inhibitor Poiypeptide The bottom non-Hpoprotein fraction obtained by ultracentrifugation for 72 hrs was analyzed for protein content by 10% SDS-polyacrylamide gel electrophoresis (LaemmH, U.K., Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4, Nature 227:680-685 (1970)), without β- mercaptoethanol pretreatment. To determine difference in small molecular weight apoHproteins, dilapidated Hpoproteins of d<1.21 g/ml from high and low HDL j baboons were separated by 15-19% SDS-polyacrylamide gel electrophoresis with -mercaptoethanol pretreatment prior to loading the samples onto the gels. xam le fi; Electroelution of Inhibitor Poiypeptide The Hpoprotein fraction of d<1.21 g/ml from high HDL j baboon plasma was dilapidated with ether-ethanol (Floren, C.H., et al., Estrogen-Induced Increase in Uptake of Cholesterol-Rich Very Low Density Lipoproteins in Perfused Rabbit Liver, MetaboHsm 30:367-374 (1981)), and separated by 15% SDS gel electrophoresis (LaemmH, (1970), supra) after addition of β-mercaptoethanol. A smaU molecular weight protein band was cut and transferred onto a tube gel (15%). A dialysis tube of molecular weight cut-off point 1000 was attached to the bottom of the gel tube to receive the electroeluted peptide. The thus electroeluted peptide was dialyzed and quantitated by comparing its absorbancy at 660 n with a known amount of stained albumin electroeluted at the same time. Example 7: Antibody Preparation The apoHpoproteins were separated by 15% SDS-gel electrophoresis and stained (LaemmH, (1970), supra). The stained bands were transferred onto a nitroceUulose membrane. The bands corresponding to the inhibitor poiypeptide were cut out (0.05 mg) and dissolved in 0.5 ml of filtered DMSO. 0.5 ml Freund's adjuvant were then added and thoroughly mixed and the mixture was injected intradermaUy into two rabbits. After 30 days, the rabbits were boosted with a simUar amount of electroeluted protein band. Antibody titer was measured by Western blotting. The rabbits were boosted again 3 times. For the preparation of antibody against the synthetic poiypeptide, 500 μg of poiypeptide were dissolved in 400 μl of titer max (CytRx Corporation, Atlanta, GA), and injected into rabbits intradermaUy. The rabbits were boosted with 500 μg of the synthetic peptide in 200 μl of Titer Max on the 28th day. The serum was tested on the 42nd day. The rabbits were bled and anti-serum was obtained as needed. Example 8: Immunoaffinity Chromatography An immunaffinity column was prepared using CnBr- activated Sepharose beads (Pharmacia Co.). The bound Hgand was IgG predpitated from the serum of rabbits having antibodies. The method used to predpitate IgG was similar to that described by McKinney and Parkinson (McKinney, M.M. and Parkinson, A., A Simple, Non-Chromatographic Procedure to Purify Immunoglobins From Serum and Ascites Fluid, J.Immunological Methods 96:271-278 (1987)). Briefly, 5 ml of rabbit serum were d uted 4-fold with acetate buffer, pH 4.0. 625 μl of capryHc acid were added dropwise to predpitate albumin and non-IgG proteins. The insoluble materials were removed by centrifugation at 10,000xg for 30 min. The supernatant was mixed with phosphate buffered saline, and the pH adjusted to 7.4 with IN sodium hydroxide. The solution was cooled to 4°C and ammonium sulfate was added to give a final concentration of 45% to precipitate the IgG. The precipitate was recovered as a peUet after centrifugation, and resuspended in phosphate buffered saline. The IgG was dialyzed overnight in 100 volumes of phosphate buffered saline, and then dissolved in sodium acetate buffer, pH 8.3, coupled to 3 g of CnBr-activated Sepharose beads, and maintained in Tris-saline, pH 7.4, until ready to use. 8 ml of plasma was incubated overnight with IgG coupled beads in Tris-saline buffered with gentle rotation. The column was then washed with Tris-saHne coupling buffer and sodium acetate buffer as described by Cheung and Albers (Cheung, M.C, and Albers, Distribution of High Density Lipoprotein Particles with Different Apolipoprotein Composition: Particles with A-I and A-II and Particles with A-I But No A-II, J. Iipid Res. 23:747-753 (1982)). The bound proteins were eluted with 0.1 M acetic acid, pH 3.0 and 1 ml aHquots were coUected and read at 280 nm to visualize the peak. The protein fraction was dialyzed immediately against phosphate buffered saline and separated by electrophoresis in 15% SDS-polyacrylamide gels. Example 9: Immunoblotting The proteins separated with SDS-polyacrylamide gels were transferred onto ImmobUon-P sheets (Millipore, Beford, MA). The sheets were incubated with antibody against inhibitor peptides after blocking of nonspecific sites. The sheets were washed and incubated again with a secondary antibody containing horseradish peroxides. The addition of boric acid buffer containing 3-amino-9- ethylcarbozole, methanol and hydrogen peroxide produced a coloration. Example 10; Amino Acid Analysis and Sequencing Stained bands of proteins were transferred onto ImmobUon-P sheets. Selected bands were cut out and hydrolyzed with 50% propionic add, 50% 12 N HC1 for 2 hrs at 135°C Amino add analysis of each sample was performed using a model 6300 amino add analyzer (Beckman Co., Palo Alto, CA) provided with System Gold software. The same bands were sequenced using a model 477A protein sequencer (AppHed Biosystems, Foster City, CA). Example 11: Preparation of Synthetic Peptides The peptides were synthesized by soHd-phase peptide synthesis as described by Barany and Merrifield (Barany, G. and Merrifield, R.B., The Peptides, Analysis, Synthesis, Biology; Gross, E. and Meinehofer, J., eds. Vol. 2, Academic Press, New York, pp. 1-284 (1980)). The 1-36 amino add synthetic peptide was assembled from the C-terminus towards the N-terminus, with the α-carboxyl group of the amino add attached to a sohd support and was then characterized by HPLC Example 12: Data Analysis The values provided in the foUowing examples are averages and are provided as mean + standard error. These values were compared using variance analysis and, if significant differences were detected, the values were compared using Duncan's Multiple Range Test (Duncan, D.B., Multiple Range and Multiple F Tests, Biometrics 11:1-12 (1955)). Example 13: Characterization of Proteins From Infranatant Fraction The CETP inhibitory activity was lost from the HDL when the Hpoproteins were extensively ultracentrifuged, e.g., for 72 hrs, or by repeated ultracentrifugation. After ultracentrifugation, the inhibitory activity was found in the infranatant fraction. To characterize the proteins, the infranatant fraction (d< 1.21 g/ml) was separated by 10% SDS polyacrylamide gel electrophoresis in the absence of -mercaptoethanol. The infranatant fraction from high HDLj baboons contained albumin, a protein sHghtly larger than apo A-I and another protein larger than apo E. Lanes A and B show nonHpoprotein fractions from high and low HDL X baboons, respectively. The protein bands, 1, 3, and 5 correspond to albumin, apo E and apo A-I, respectively. The protein bands 2 and 4 of molecular weights between 1 and 3, and 3 and 5, respectively, correspond to proteins with molecular weights of 41,000 and 31,000, respectively. Protein samples from high HDL X baboons show only bands corresponding to albumin, a protein of molecular weight 41,000 and a protein of molecular weight 31,000. The molecular weights were determined with standard proteins separated on similar gels (gel picture not shown). The infranatant fraction from low HDL j baboons contains these proteins as weU, but in addition, it also contains apo A-I and apo E. Both proteins in the apo A-I region were identified by immunoblotting with antibody to apo A-I. Similarly, both proteins in the apo E region were identified by immunoblotting with antibody to apo E. The molecular weights of the proteins detected by immunoblotting with apo A-I and apo E show a difference of about 4kD (picture not shown). The estimated molecular weight of apo A-I is about 27,500 and that of the modified apo A-I is about 31,000. SimUarly, the estimated molecular weight of apo E is about 37,000 and that of modified apo E is about 41,000. Both apo A-I and apo E are modified by a protein of about 4,000 molecular weight. Example 14: Detection of CETP Inhibitor Peptide in Plasma of High HDL, Baboons To determine if a common poiypeptide of molecular weight 4,000 was modifying both apo A-I and apo E, plasma Hpoproteins of d < 1.21 g/ml were separated from high and low HDL j baboons by 18% SDS-polyacrylamide gel electrophoresis with -mercaptoethanol. A higher amount of a 4kD protein was detected in Hpoprotein from high HDL j baboons as compared to Hpoprotein from low HDL j baboons (gel picture not shown). To determine if the 4kD poiypeptide inhibits CE transfer, the poiypeptide and albumin were electroluted from the gels, and used in increasing concentrations in a CE transfer assay mixture with Hpoproteins from low HDL X baboons as described in Example 4 above. Albumin had no effect on the transfer of CE from HDL to VLDL+LDL. The 4kD poiypeptide, on the other hand, significantly inhibited CETP activity. (Results are not shown). Example 15: Characterization of Poiypeptide by Affinity Chromatography. Rabbit antibody was prepared against the 4kD poiypeptide isolated from Hpoproteins obtained from high HDL j baboons. This antibody was used to prepare an immunoarfinity column. The Hpoproteins of d< 1.21 g/ml were passed over the immunoaffinity column. The bound Hpoproteins eluted with 0.1 M acetic acid and separated in 15% SDS-polyacrylamide reducing gels. 4kD, and 31kD polypeptides, and a minor band corresponding to a 41kD poiypeptide were detected. The column bound peptides (100 μg) inhibited by 31.3 ± 1.4% the transfer of CE (mean ± SE, n=3) in the CETP assay from low HDL j baboons. On the other hand, the addition of IgG to the assay from high HDL X baboons increased CE transfer by 44.3 + 1.5% (n=3), but had no effect on CE transfer from low HDL j baboons. Example I fi; Comparison of 4kD Inhibitor Poiypeptide with Sequence Data Bank The sequence of this poiypeptide was compared to sequence of known proteins using Sequence Data Bank (Reardon, W. R. and Iipman, D.J., PNAS (USA) 85:2444-2448 (1988)), and was found to have 100% homology with human and crab-eating macaque apo C-I. The sequence was then compared with apo C-I from baboons and found to be 100% homologous (private communication from Dr. Hixson of the Southwest Foundation for Biomedical Research). Example 17: Characterization of Inhibitor Poiypeptide with Synthetic Peptides Based on its molecular weight, it was determined that the 4kD poiypeptide contained approximately 36 amino adds. Three peptides were synthesized beginning from the C- terminal end of the apo C-I sequence. The first peptide contained 9 amino adds, the second peptide contained 21 amino adds and the third peptide contained 36 amino adds. The 36 amino acid peptide had an amino add sequence similar to the 4,000 MW poiypeptide, the other two were fragments of the synthetic peptide starting from its C-terminus. 50 μg of these peptides were used in a CETP assay with Hpoproteins of the low HDL j baboons as described in Example 4 above. A soluble heHcal peptide with a 1,900 molecular weight utilized as control. The 36 amino add poiypeptide significantly (p< 0.01) inhibited CE transfer from HDL to VLDL and LDL, whUe the others, including the control peptide, did not. Example 18: Antibody Against 36 Amino . Acid Inhibitor Peptide Antibody against the 36 amino acids inhibitor peptide was prepared in rabbits as described in Example 7, and used for immunoblotting. The thus prepared antibody recognized the 4kD peptide as weU as a 31kD poiypeptide from Hpoproteins of high HDL j baboons. To determine if both the apo C-I and the 4kD poiypeptide were present in the plasma of high and low HDL j baboons, Hpoproteins from both phenotypes were separated by 10% SDS gel electrophoresis and immuno-blotted. Two protein bands were detected with the antibody by immunoblotting of samples from high HDL j baboons. Only a single band was detected in samples from low HDL j baboons. In addition, isoelectric focusing patterns of the synthetic peptide suggest that the peptide is a sHghtly basic protein. Example 19: CETP Inhibition by Various Peptide Fragments A CE transfer assay as described in Example 4 above was conducted using HDL ! baboon plasma. 3 H HDL and the VLDL+LDL carriers, in the presence of CETP enzyme to mediate the exchange. The reactions were conducted at 37°C and 4°C (control) in dupHcate. The results are shown in Table 1 below. TABLE 1: CETP INHIBITION BY VARIOUS PEPTIDE FRAGMENTS CETP [H 3 ] HDL SYNTHETIC VLDL+LDL TEMP. INHIBITION PEPTIDE (μl) (μg) (μg) (μg) (°C) (%) 125 100 300 37 0 125 100 300 4 125 100 50 300 37 0 y ammo aciα 125 100 300 4 125 100 ,50 300 37 4 21 ammo acid 125 100 300 4 125 100 50 300 37 32 36 amino acid 125 100 300 4 125 100 50 300 37 0 9 ammo acid 125 100 300 4 125 100 50 300 37 26 21 ammo acid 125 100 300 4 125 100 50 300 37 32 36 ammo acid 125 100 300 4 Example 20: Inhibition of CETP From Humans by Synthetic CETP Inhibitor Peptide. Cholesteryl ester transfer activity from human plasma was assayed by the procedure described by the inventors (Kushwaha R.S., Rainwater D.L., WilHams M.C, Getz G.S., and McGiU H.C., Jr., Impaired Plasma Cholesteryl Ester Transfer with Accumulation of Large High Density Lipoproteins in Some Families of Baboons (Papio sp.). J. Iipid Res. 31:965-973, 1990). In short, [ 3 H] cholesteryl ester- labeled HDL (10-μg of cholesteryl esters with a specific activity of 3-4 x 10 6 dpm/mg cholesteryl ester) from low HDL j baboons was incubated with 50-100 μg of VLDL+LDL cholesteryl ester from baboons. The incubations were carried out in the presence of 100 μl of Hpoprotein defident serum (LPDS) obtained from humans and 2mM DTNB. The total volume of the assay was 1 ml. The incubations were carried out for 1-2 h at 4° and 37°. At the end of the incubation, 40 μl of heparin (5000 units /ml), 0.5 ml of plasma, and 60 μl of 1 M MnCl 2 were added in that order. The mixture was vortexed, incubated for 0.5 h on ice, and centrifuged for 10 minutes. The radioactivity was measured in the supernatant fraction by sdntiUation spectrometry. The difference between 4° and 37° was considered to reflect the CETP -mediated transfer. At the same time each set of human CETP incubations were run in the presence of synthetic CETP inhibitor peptide (baboon apo C-I terminal peptide with 38 amino adds, Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu- Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO. 2) and the transfer of cholesteryl ester from HDL to VLDL+LDL was determined in the presence of CETP inhibitor. In some cases synthetic CETP inhibitor was similar to human apo C-I terminal peptide with 38 amino acids (Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp- Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu- Leu-He-Ser-Arg-Ue-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO. 3). The results of these experiments are provided in the foUowing tables: TABLE 2. Inhibition of Hainan CETP by CETP Inhibitor peptide (Baboon sequence). S.N. Source of I * Η] HDL Inhibitor VLDL+LDL Temp. CETP Inhibition human CETP (μg) peptide (μg) fO (μl) (H) (μg) SH 10 00 60 4 200 10 00 60 37 200 00 10 100 60 4 200 RK 10 00 50 4 200 10 00 60 37 200 00 10 100 60 4 200 10 100 60 87 200 72 EJ 10 00 60 4 200 10 00 60 37 200 00 10 100 60 4 200 10 100 60 37 200 85 GL 10 00 60 4 200 10 00 60 37 200 CETP 10 100 50 4 200 NOT 10 100 50 37 200 ACTIVE KR 10 00 50 4 200 10 00 50 37 200 00 10 100 60 4 200 10 100 60 37 200 82 AB 10 00 50 4 200 10 00 60 37 200 00 10 100 60 4 200 10 100 50 37 200 60 GL 10 00 50 4 200 10 00 60 37 200 00 10 100 60 4 200 10 100 50 37 200 85 KC 10 00 50 4 200 10 00 50 37 200 00 10 100 50 4 200 10 100 50 37 200 83 TH 10 00 50 4 200 10 00 50 37 200 00 10 100 50 4 200 10 100 60 37 200 86 UM 10 00 60 4 200 10 00 50 37 200 00 10 100 50 4 200 10 EW 10 00 50 4 200 10 00 60 37 200 10 100 50 4 200 10 100 50 37 200 71 12. ML 10 00 60 4 200 10 00 60 37 200 10 100 50 4 200 10 100 50 37 200 77 TABLE S. Comparisons of CETP inhibitor peptide from human and baboons on CETP transfer in baboons. [ d H] HDL Inhibitor Inhibitor VLDL+LDL Teπφ. CETP Inhibition (μg) peptide peptide (μg) ( β Q (μl) (%) (baboon) (human) (μg) (μg) 10 00 00 50 4 100 10 00 00 50 37 100 00 10 50 00 50 4 100 10 50 00 50 37 100 52 10 00 00 50 4 100 10 00 00 50 37 100 00 10 00 50 50 4 100 10 00 50 50 37 100 60 10 100 60 37 200 90 EW 10 00 60 4 200 10 00 60 37 200 00 10 100 60 4 200 10 100 60 37 200 12. ML 10 00 60 4 200 10 00 50 37 200 10 100 60 4 200 10 100 60 37 200 TABLE 3. Comparisons of CETP inhibitor peptide from human and baboons on CETP transfer in baboons. [ 3 H] HDL Inhibitor Inhibitor VLDL+LDL Temp. CETP Inhibition (μg) peptide peptide (μg) ( β C) (μl) (%) (baboon) (human) (μg) (μg) 10 00 00 50 4 100 10 00 00 50 37 100 00 10 50 00 50 4 100 10 50 00 50 37 100 52 10 00 00 50 4 100 10 00 00 50 37 100 00 10 00 50 50 4 100 10 00 50 50 37 100 60 Example 21: Method of preparing [ 3 H] HDL Cholesteryl estei labeled HDL, VLDL+LDL LPDS (human). [ 3 H] cholesteryl ester-labeled HDL and acceptor hpoproteins (d < 1.045 g/ml) were prepared as described by the inventors (Kushwaha, et al. J. Lipid Res. 31:965-973, 1990). Low HDL, baboons were bled after immobilization with ketamine HC1 (10 mg/kg). Blood was coUected in tubes containing EDTA (1 mg/ml) and plasma separated by low speed centrifugation at 6°C. Plasma was immediately treated with sodium azide (0.2 gΛ), gentamycin sulfate (0.1 g/1), chloramphenicol (0.05 gΛ). and phenylmethylsulfonyl fluoride (0.5 mM). Tritiated cholesteryl oleate (20 μCi/ml) dissolved in ethanol was added to the plasma. The plasma was flushed with nitrogen and incubated for 20 h at 4°C. After incubation, HDL was isolated by density gradient ultracentrifugation (Kushwaha, et al., 1990), 32A dialyzed against normal saline/EDTA (0.00 IM) and used as a substrate for cholesteryl ester transfer reaction. VLDL+LDL (d < 1.045 g/ml) and LPDS were isolated by sequential ultracentrifugation from 100-200 ml blood obtained from two to four SUBSTIT 33 baboons as described by the inventors (Kushwaha, et al., 1990). Total and free cholesterol and HDL and VLDL+LDL were measured by enzymatic assay (Wako Pure Chemical Co.). Experiment 22: Method of isolating human CETP To isolate human CETP, blood (5-10 ml) from human subjects was obtained in tubes containing EDTA (1 mg/ml). Plasma was obtained by low speed centrifugation. Plasma was kept on ice and the LDL was precipitated by adding 40 μl heparin/ml (5000 units /ml) of plasma along with 60 μl/ml of plasma of IM MnCl 2 . The plasma was vortexed and incubated on ice for 15 min. FoUowing incubation, the supernatant was recovered by centrifugation. The procedure was repeated twice to completely precipitate VLDL+LDL in the plasma. Afterwards, 80 μl/ml of 10% dextran sulfate per ml was added to the supernatant and incubated for 15 min. The mixture was centrifuged and the supernatant was coUected and used as the source of CETP. 34 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: KUSHWAHA, RAMPRATAP McGILL JR., HENRY C. KANDA, PATRICK (ii) TITLE OF INVENTION: CETP INHIBITOR POLYPEPTIDE, ANTIBODIES AGAINST THE SYNTHETIC POLYPEPTIDE, AND PROPHYLACTIC AND THERAPEUTIC ANTI-ATHEROSCLEROSIS TREATMENTS. (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: COX & SMITH INCORPORATED (B) STREET: 112 EAST PECAN STREET, SUITE 1800 (C) CITY: SAN ANTONIO (D) STATE: TEXAS (E) COUNTRY: US (F) ZIP: 78205 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: HAYMOND, W. B. (B) REGISTRATION NUMBER: 35,186 (C) REFERENCE/DOCKET NUMBER: S-0072.187 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 210-554-5260 (B) TELEFAX: 210-226-8395 (C) TELEX: 767609 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 amino acids (B) TYPE: amino acid (C) STRANDEDNΞSS: single (D) TOPOLOGY: linear 35 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Asp Val Ser Ser Ala Leu Asp Lys Leu Lys Glu Phe Gly Asn Thr Leu 1 5 10 15 Glu Asp Lys Ala Trp Glu Val lie Asn Arg lie Lys Gin Ser Glu Phe 20 25 30 Pro Ala Lys Thr 35 (3) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Ala Pro Asp Val Ser Ser Ala Leu Asp Lys Leu Lys Glu Phe Gly Asn 1 5 10 15 Thr Leu Glu Asp Lys Ala Trp Glu Val lie Asn Arg lie Lys Gin Ser 20 25 30 Glu Phe Pro Ala Lys Thr 35 (4) INFORMATION FOR SEQ ID NO:3i (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Thr Pro Asp Val Ser Ser Ala Leu Asp Lys Leu Lys Glu Phe Gly Asn 36 1 5 10 15 Thr Leu Glu Asp Lys Ala Arg Glu Leu lie Ser Arg lie Lys Gin Ser 20 25 30 Glu Leu Ser Ala Lys Met 35

Claims: 37 CLAIMS 1. A substantially pure poiypeptide having activity inhibitory of cholesteryl ester transfer protein comprising at least a part of peptides selected from the group consisting of: Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-fle-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys- Thr (SEQ. ID. NO: 2) and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys- Met (SEQ. ID. NO: 3), 2. The cholesteryl ester transfer protein inhibitory poiypeptide of claim 1, in freeze dried form. 3. An anti- atherosclerosis composition, comprising an anti- athero-sclerosis effective amount of at least a part of a poiypeptide selected from the group consisting of: Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-ϋe-Asn-Arg-πe-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys- Thr (SEQ. ID. NO: 2) and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys- Met 38 (SEQ. ID. NO: 3), and a pharmaceutically-acceptable carrier. 4. An anti-atherosclerosis kit, comprising in separate sterile containers at least 10 to 400 mg of the composition of claim 3; at least one syringe; and at least one needle. 5. An antibody having specificity for a poiypeptide comprising at least a part of a poiypeptide selected from the group consisting of: Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys- Thr (SEQ. ID. NO: 2) and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-He-Ser-Arg-ϋe-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys- Met (SEQ. ID. NO: 3), 6. The antibody of claim 5, being a polyclonal antibody. 7. The antibody of claim 5, being capable of specifically binding to modified apo A-I. 8. The antibody of claim 5, being capable of specifically binding to the baboon CETP inhibitor poiypeptide. 9. The antibody of claim 5, being capable of specifically binding to modified apo E. 10. The antibody of claim 5, being capable of specifically binding to apo C-I. 11. A method of preventing atherosclerosis in a mammal being predisposed to that condition, comprising administering to the 39 mammal a prophylactically effective amount of a poiypeptide comprising at least a part of a peptide selected from the group consisting of: Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys- Thr (SEQ. ID. NO: 2) and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-ϋe-Ser-Arg-ϋe-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys- Met (SEQ. ID. NO: 3), 12. The method of claim 11, wherein the poiypeptide is administered in an amount of about 10 to 200 mg. 13. The method of claim 22, wherein the poiypeptide is administered intravenously. 14. The method of claim 22, wherein the mammal is a human. 15. A method of treating a mammal afflicted with atherosclerosis comprising administering to the mammal a therapeutically effective amount of a poiypeptide comprising at least one part of a peptide selected from the group consisting of: Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-πe-Asn-Arg-ϋe-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys- Thr (SEQ. ID. NO: 2) and 40 Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys- Met (SEQ. ID. NO: 3). 16. The method of claim 15, wherein the poiypeptide is administered in an amount of about 10 to 400 mg. 17. The method of claim 15, wherein the poiypeptide is administered intravenously. 18. The ethod of claim 15, wherein the mammal is a human. IN THE INTERNATIONAL BUREAU OF WIPO APPLICATION SERIAL NO. § ATTY DOCKET NO. S-0072.199 PCT/US94/08624 § § APPLICANT: Southwest § PCT FILING DATE: 02 Aug. 94 Foundation For Biomedical § Research § § TITLE: CETP INHIBITOR POLYPEPTIDE ANTIBODIES AGAINST THE SYNTHETIC POLYPEPTIDE AND PROPHYLACTIC AND THERAPEUTIC ANTI-ATHEROSCLEROSIS TREATMENTS REQUEST FOR RECTIFICATION UNDER RULES 91 AND 91.1(bMcϊ The International Bureau of WIPO 34 chemin des Colombettes 1211 Geneva 20 Switzerland Sir: This is a Request For Rectification Under Rules 91 and Rule 91.1(b)(c) to correct an obvious error transmitted in the above-referenced PCT application as originally filed. On 21 November 1994, Applicant submitted a Request under Rules 91 and 91.1(b)(c) to correct an obvious error. However, on 05 January 1995, the International Searching Authority refused to authorize the rectification and a copy of that refusal in enclosed herewith. Applicant hereby resubmits the Request For Rectification Under Rules 91 and 91.1(b)(c) to the International Bureau of WIPO for consideration and acceptance. Enclosed are Replacement Pages 6 and 6a, a copy of the Notification of Decision Concerning Request for Rectification, and a check in the amount of $50.00 to cover the fees incurred due to the filing of this Request. If the fee is deficient, the International Bureau is requested to contact the undersigned attorney for Applicant, and any deficiency will be remitted promptly. REMARKS Applicant filed the above-referenced PCT application on 02 August 1994. Applicant recently discovered a brief omission on page 6 between lines 10-11. It is obvious when reading lines 8-10 on page 6 that a schematic figure is missing and such omission is clearly rectifiable with the submission of Replacement pages 6 and 6a and such action is earnestly solicited. Respectfully submitted, Reg. No. 35.186 Cox & Smith Incorporated 112 East Pecan Street, Suite 1800 San Antonio, Texas 78205-1536 (210) 554-5260 (210) 226-8395 (FAX) ATTORNEYS FOR APPLICANT I hereby certify that the foregoing document with Replacement Pages 6 and 6A, the Notification of Decision Concerning Request for Rectification are being faxed to the International Bureau of WIPO on this date. Replacement Page 6 Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following discussion. Description of the Preferred Embodiment This invention arose from the desire by the inventors to provide a novel and unobvious approach to the prevention and treatment of atherosclerosis in humans. A schematic of what is known about the association of cholesterol with the different fractions of lipoproteins in plasma and in the liver is shown below: The metabolic steps leading to the accumulation ( ) of HDL^ VLDL and LDL are shown above. HDL 2+3 collects cholesterol from extrahepatic cells, which is then esterified by LCAT to form cholesteryl esters (CE) and stored in the core of the particles. The HDL becomes larger in size (HDL^ and may pick up apo E to attain a particle which is removed from LDL receptors (LDL-R) on liver cells. The CE enriched HDL l may also donate CE to VLDL and LDL. This is mediated by CETP. Due to the presence of the CETP inhibitor, such as the one provided herein, CE transfer is slow (bar) and the reciprocal transfer of triglycerides (TG) does not take place (X). The triglyceride-poor HDL is thus not a suitable substrate for Replacement Page 6A hepatic triglyceride lipase (HTGL). Due to the presence of the CETP inhibitor, in plasma, VLDL and LDL are thus not available to the liver. As a consequence of this, the Hver then increases the expression of messages for the increased production of LDL receptor and 3 hydroxy, methyl, glutaryl-coenzyme A (HMG-COA) synthase. The increase in LDL receptor in the Hver leads to an increase in uptake of LDL or HDL, with apo E, and consequently, to a greater deHvery of cholesterol to the Hver. An increase in synthesis of HMG- CoA synthase leads to an increase in synthesis of cholesterol in the Hver to meet aU cellular needs. Thus, the presence of a CETP inhibitor in plasma will prevent the uptake of VLDL and/or LDL by tissues as weU as the deposition of cholesteryl esters. In general, high levels of HDL have an anti-atherosclerogenic effect whereas high levels of LDL have an atherogenic effect. The

Inventors: KANDA PATRICK, KUSHWAHA RAMPRATAP S, MCGILL HENRY C JR, KANDA, PATRICK, KUSHWAHA, RAMPRATAP, S., MCGILL, HENRY, C., JR.
Applicants: SOUTHWEST FOUND BIOMED RES, SOUTHWEST FOUNDATION FOR BIOMEDICAL RESEARCH
Application year: 1994
Main classification: C07K14
IPC codes: A61K38, A61K39, A61P9, C07K14, C07K16